Yamada Shinya, Khankaldyyan Vazgen, Bu Xiang, Suzuki Atsushi, Gonzalez-Gomez Ignacio, Takahashi Kouichi, McComb J Gordon, Laug Walter E
Department of Neurosurgery, Tokai University Oiso Hospital, Japan.
Tokai J Exp Clin Med. 2004 Dec;29(4):167-73.
To improve currently used techniques to implant tumor cells into the parenchyma of the mouse brain.
The stereotactic injection of 0.5 to 5 microl of indigo carmine over 5 to 40 minutes into the caudate/putamen nuclei of the mouse was done followed by sacrifice and examination of the brain injection site. 1 microl containing 10(5) U87MG glioma cells were stereotactically implanted into the caudate/ putamen nuclei over 20 minutes. The animals were sacrificed from one hour to 63 days after implantation and the brain examined and tumor size measured.
An injection of 1 microl of indigo carmine over 20 minutes produced a spherical deposit of dye within the caudate/putamen nuclei. Larger volumes of indigo carmine or shorter injection times resulted in dye spreading along the injection tract or into the ventricles or subarachnoid space. Using the results of the dye studies, the same parameters were used to successfully inject and confine the glioma cells to the caudate/putamen nuclei in 30 of 32 mice. No tumor was found in 2 animals and appears to be explained by obstruction of the injection cannula. The tumor cells appeared viable an hour after injection. However by day three, considerable necrosis of tumor cells were noted, the effects of which resolved by day five. On day six, the injection site was comparable to that at one hour. In the early phase, until the fifth week, tumor volume doubling time was ten days while afterward it was only five days.
The technique described allows the highly accurate and reproducible introduction of a given number of cells into a specific area of the mouse brain. This should reduce the intragroup variability, be it control or therapeutic, allowing better assessment of outcome with fewer number of mice.
改进目前将肿瘤细胞植入小鼠脑实质的技术。
在5至40分钟内将0.5至5微升靛胭脂立体定向注射到小鼠的尾状核/壳核，随后处死小鼠并检查脑内注射部位。将含有10(5)个U87MG胶质瘤细胞的1微升溶液在20分钟内立体定向植入尾状核/壳核。在植入后1小时至63天处死动物，检查脑部并测量肿瘤大小。
在20分钟内注射1微升靛胭脂会在尾状核/壳核内产生球形染料沉积。注射更大体积的靛胭脂或更短的注射时间会导致染料沿注射通道扩散或进入脑室或蛛网膜下腔。利用染料研究的结果，采用相同参数成功地将胶质瘤细胞注射并限制在32只小鼠中的30只的尾状核/壳核内。2只动物未发现肿瘤，似乎是由于注射套管堵塞所致。注射后1小时肿瘤细胞看起来是活的。然而到第3天，注意到肿瘤细胞有相当程度的坏死，其影响在第5天消退。在第6天，注射部位与1小时时相当。在早期，直到第5周，肿瘤体积倍增时间为10天，而之后仅为5天。
所描述的技术能够将给定数量的细胞高度准确且可重复地引入小鼠脑的特定区域。这应能减少组内变异性，无论是对照还是治疗方面，从而用更少数量的小鼠更好地评估结果。
